The present invention relates to products and methods useful for the production of a single-vial lyophilized nucleic acid/formulating agent complex with favorable physical and dosage characteristics.
None of the information provided herein is admitted to be prior art to the present invention, but is provided only to aid the understanding of the reader.
Gene therapy has become a major area of research in rug development. More practical and effective gene delivery methods continue to aid the advancement of the clinical and/or commercial uses of gene therapy, which generally are expected to deliver the product to the targeted cells in sufficient quantities.
In the past, incorporation of nucleic acid into a conventional dosage form has been a challenge due to the chemical degradation or physical aggregation of the nucleic acid prior to administration. The preferred method of administration of a nucleic acid complex is in a liquid form, so past methods have included storing the nucleic acid in a separate vial and combining the necessary components shortly prior to administration. This was a generally effective method, however it was relatively expensive and difficult to prepare and produced a rather unstable product which was somewhat difficult to administer. The stabilization of polynucleotide complexes by adding a cryoprotectant compound and lyophilizing the resulting formulation is described in Szoka et al., International Patent Publication No. WO 96/41873, published Dec. 27, 1996, entitled xe2x80x9cDry Powder Formulations of Polynucleotide Complexesxe2x80x9d, incorporated herein by reference in its entirety, including any drawings.
In prior attempts to make nucleic acid formulations, the method of mixing has typically been a conventional, slow (but not controlled) mixing of the DNA complex and formulating agent. The result was a non-homogenous complex with particles of relatively large size (approximately 150 nanometers). For ease of administration of the DNA complex, it would be desirable to have a smaller and more uniform particle size. Thus, despite the above, there remains a need for a single-vial homogenized nucleic acid formulation of a relatively small and uniform particle size, especially one protected from degradation and with an increased ability to transfect cells relative to the non-formulated nucleic acid, as well as easier methods of preparation and storage.
This invention features compositions and methods for a cost-effective production of isolated, enriched or purified nucleic acids, preferably DNA plasmid, in a conventional dosage form without significant reduction of its biological activity. Thus, the present invention provides a single-vial, homogenized complex created by in-line mixing, where the resulting complex is lyophilized for storage and re-hydrated for administration. The in-line mixing preferably utilizes two tubes which lead into a mixer and which will serve to produce a mixture of a nucleic acid and a formulating agent. The complex in this invention, though formulated in a single vial, maintains favorable physical characteristics and potency. The use of this single vial formulation will result in: (1) reduced manufacturing costs, (2) ease of manufacturing and quality control testing, (3) product stability, and (4) increased doctor/patient compliance and relative ease of administration. These attributes, and the details that follow, provide advantages over the previously used formulations.
Thus, in one aspect, the invention features an in-line mixer containing a confined flowing system of isolated, enriched or purified liquid nucleic acid molecules.
By an xe2x80x9cin-line mixerxe2x80x9d is meant a device through which liquids to be contacted with one another are passed and which is used for continuous or semibatch operations. The mixer may include tubing and together with the liquid forms a confined flowing system. The volume of liquid in the mixer is limited only by the size and shape of the mixer. The mixers may utilize mechanical agitation, but when mechanical agitation is not used, other methods such as jet mixers, injectors, orifices, mixing nozzles, valves, and pumps may be used. Many in-line mixers are used commercially in chemical and chemical engineering applications, such as in the treatment of petroleum distillates, in vegetable oil refining, in some metal extractions and other applications. Because many types of these mixers are commercially available for use in chemistry and chemical engineering, those skilled in the art could easily design and make other in-line mixers with minor modifications that would still be suitable for treating nucleic acids as described herein.
The in-line mixer preferably is made up of two inlets in a Y-shaped configuration which join at an intersection to form a single outlet. In one embodiment of the invention, the in-line mixer includes a static mixer after the Y-shaped intersection.
By xe2x80x9cnucleic acid moleculesxe2x80x9d, it is meant polynucleotides, i.e., a polymer of deoxyribonucleotides (DNA) or ribonucleotides (RNA) which includes naked DNA, a nucleic acid cassette, naked RNA, nucleic acid contained in vectors or viruses, both RNA and DNA including: cDNA, genomic DNA, plasmid DNA or condensed nucleic acid, nucleic acid formulated with cationic lipids, nucleic acid formulated with peptides, antisense molecules, cationic substances, RNA or mRNA. Examples of suitable nucleic acid molecules include those described in Szoka et al., International Application xe2x80x9cDry Powder Formulations of Polynucleotide Complexesxe2x80x9d, International Patent Publication No. WO 96/41873, published Dec. 27, 1996, and Rolland, et al., International Patent Publication WO 96/21470, published Jul. 18, 1996, entitled, xe2x80x9cFormulated Nucleic Acid Compositions and Methods of Administering the Same for Gene Therapyxe2x80x9d which are incorporated herein by reference in their entirety, including any drawings. These are only examples and are not meant to be limiting. Additionally the nucleic acid molecules may be one or more plasmids with a eukaryotic promoter that expresses one or more therapeutic molecules. The nucleic acid molecules are, in certain aspects and embodiments, isolated, purified or enriched, as defined below in the Detailed Description at Section I.D.
The nucleic acid molecule may be isolated from a natural source by cDNA cloning or subtractive hybridization or synthesized manually. The nucleic acid molecule may be synthesized manually by the triester synthetic method or by using an automated DNA synthesizer.
The term xe2x80x9ccDNA cloningxe2x80x9d refers to hybridizing a small nucleic acid molecule, a probe, to genomic cDNA. The probe hybridizes (binds) to complementary sequences of cDNA.
The term xe2x80x9ccomplementaryxe2x80x9d describes two nucleotides that can form multiple favorable interactions with one another. For example, adenine is complementary to thymine as they can form two hydrogen bonds. Similarly, guanine and cytosine are complementary since they can form three hydrogen bonds. Thus if a nucleic acid sequence contains the following sequence of bases, thymine, adenine, guanine and cytosine, a xe2x80x9ccomplementxe2x80x9d of this nucleic acid molecule would be a molecule containing adenine in the place of thymine, thymine in the place of adenine, cytosine in the place of guanine, and guanine in the place of cytosine. Because the complement can contain a nucleic acid sequence that forms optimal interactions with the parent nucleic acid molecule, such a complement can bind with high affinity to its parent molecule.
In a preferred embodiment, the nucleic acid administered is plasmid DNA which includes a xe2x80x9cvectorxe2x80x9d. The nucleic acid can be, but is not limited to, a plasmid DNA vector with a eukaryotic promoter which expresses a protein with potential therapeutic action, such as, for example; hGH, VEGF, EPO, IGF-1, TPO, Factor IX, IFN-xcex1, IFN-xcex2, IL-2, IL-12, or the like.
As used herein, the term xe2x80x9cplasmidxe2x80x9d refers to a construct made up of genetic material (i.e., nucleic acids). It includes genetic elements arranged such that an inserted coding sequence can be transcribed in eukaryotic cells. Thus, the plasmid preferably is an extrachromosomal genetic element consisting of a circular duplex of DNA which can replicate independently of chromosomal DNA. Plasmids are used in gene transfer, as the vehicle by means of which DNA fragments can be introduced into a host organism, and are associated with the transfer of antibiotic resistance.
The term xe2x80x9cvectorxe2x80x9d as used herein refers to a construction comprised of genetic material designed to direct transformation of a targeted cell. A vector contains multiple genetic material, preferably contiguous fragments of DNA or RNA, e.g., DNA derived from a plasmid, cosmid, phasmid or bacteriophage or synthesized by chemical or enzymatic means, positionally and sequentially oriented with other necessary elements such that the nucleic acid can be transcribed and when necessary translated in the transfected cells. The vector can contain one or more unique restriction sites for this purpose, and may be capable of autonomous replication in a defined host or organism such that the cloned sequence is reproduced. The vector may have a linear, circular, or supercoiled configuration and may be complexed with other vectors or other materials for certain purposes. The components of a vector can include but are not limited to a DNA molecule incorporating: (1) a sequence encoding a therapeutic or desired product; and (2) regulatory elements for transcription, translation, RNA stability and replication.
In the present invention the preferred vector comprises the following elements linked sequentially at an appropriate distance to allow functional expression: a promoter, a 5xe2x80x2 mRNA leader sequence, a translation initiation site, a nucleic acid cassette containing the sequence to be expressed, a 3xe2x80x2 mRNA untranslated region, and a polyadenylation signal sequence. As used herein the term xe2x80x9cexpression vectorxe2x80x9d refers to a DNA vector that contains all of the information necessary to produce a recombinant protein in a heterologous cell.
In addition, the term xe2x80x9cvectorxe2x80x9d as used herein can also include viral vectors, although non-viral vectors are preferred. A xe2x80x9cviral vectorxe2x80x9d in this sense is one that is physically incorporated in a viral particle by the inclusion of a portion of a viral genome within the vector, e.g., a packaging signal, and is not merely DNA or a located gene taken from a portion of a viral nucleic acid. Thus, while a portion of a viral genome can be present in a vector of the present invention, that portion does not cause incorporation of the vector into a viral particle and thus is unable to produce an infective viral particle.
A vector as used herein can also include DNA sequence elements which enable extra-chromosomal (episbmal) replication of the DNA. Vectors capable of episomal replication are maintained as extra-chromosomal molecules and can replicate. These vectors are not eliminated by simple degradation but continue to be copied. These elements may be derived from a viral or mammalian genome. These provide prolonged or xe2x80x9cpersistentxe2x80x9d expression as described below.
The term xe2x80x9cpersistent expressionxe2x80x9d as used herein refers to introduction of genes into the cell together with genetic elements which enable episomal (i.e., extrachromosomal) replication. This can lead to apparently stable transformation of the cell without the integration of the novel genetic material into the chromosome of the host cell.
xe2x80x9cStable expressionxe2x80x9d as used herein relates to the integration of genetic material into chromosomes of the targeted cell where it becomes a permanent component of the genetic material in that cell. Gene expression after stable integration can permanently alter the characteristics of the cell and its progeny arising by replication leading to stable transformation.
The vector can be used to provide expression of a nucleic acid sequence in tissue. In the present invention this expression preferably is enhanced by providing stability to an mRNA transcript from the nucleic acid sequence and/or secretion of the therapeutic protein. Expression includes the efficient transcription of an inserted gene or nucleic acid sequence within the vector. Expression products may be proteins including but not: limited to pure protein (polypeptide), glycoprotein, lipoprotein, phosphoprotein, or nucleoprotein. Expression products may also be RNA. The nucleic acid sequence is contained in a nucleic acid cassette. Expression of the nucleic acid can be continuous or controlled by endogenous or exogenous stimuli.
The term xe2x80x9ccontrolxe2x80x9d or xe2x80x9ccontrolledxe2x80x9d as used herein relates to the expression of gene products (protein or RNA) at sufficiently high levels such that a therapeutic effect is obtained. Levels that are sufficient for therapeutic effect are lower than the toxic levels. Levels of expression for therapeutic effect within selected tissues corresponds to reproducible kinetics of uptake, elimination from cell, post-translational processing, and levels of gene expression, and, in certain instances, regulated expression in response to certain endogenous or exogenous stimuli (e.g., hormones, drugs).
The term xe2x80x9cnucleic acid cassettexe2x80x9d as used herein refers to the genetic material of interest which codes for a protein or RNA. The nucleic acid cassette is positionally and sequentially oriented within the vector such that the nucleic acid in the cassette can be transcribed into RNA, and when necessary, translated into a protein in the transformed tissue or cell. Preferably, the cassette has 3xe2x80x2 and 5xe2x80x2 ends adapted for ready insertion into a vector, e.g., it has restriction endonuclease sites at each end.
The term xe2x80x9ctissuexe2x80x9d as used herein refers to a collection of cells specialized to perform a particular function or can include a single cell. The cells may be of the same type or of different types.
In preferred embodiments, the nucleic acid contains a coding region transcriptionally linked to a transcriptional control sequence.
In this context, xe2x80x9ctranscriptionally linkedxe2x80x9d means that in a system suitable for transcription, transcription will initiate under the direction of the control sequence(s) and proceed through sequences which are transcriptionally linked with that control sequence(s). Preferably no mutation is created in the resulting transcript, which would alter the resulting translation product.
The term xe2x80x9ccoding regionxe2x80x9d or xe2x80x9ccoding sequencexe2x80x9d refers to a nucleic acid sequence which encodes a particular gene product for which expression is desired, according to the normal base pairing and codon usage relationships. Thus, the coding sequence must be placed in such relationship to transcriptional control sequences (possibly including control elements and translational initiation and termination codons) that a proper length transcript will be produced and will result in translation in the appropriate reading frame to produce a functional desired product.
The term xe2x80x9ctranscriptional control sequencexe2x80x9d refers to sequences which control the rate of transcription of a transcriptionally linked coding region. Thus, the term can include elements such as promoters, operators, and enhancers. For a particular transcription unit, the transcriptional control sequences will include at least a promoter sequence.
The plasmid, in preferred embodiments, may also contain a growth hormone 3xe2x80x2 untranslated region, preferably from a human growth hormone gene.
A xe2x80x9cgrowth hormone 3xe2x80x2 untranslated regionxe2x80x9d is a sequence located downstream (i.e., 3xe2x80x2) of the region encoding material polypeptide and including at least part of the sequence of the natural 3xe2x80x2 UTR/poly(a) signal from a growth hormone gene, preferably the human growth hormone gene. This region is generally transcribed but not translated. For expression in eukaryotic cells it is generally preferable to include sequence which signals the addition of a poly-A tail. As with other synthetic genetic elements a synthetic 3xe2x80x2 UTR/poly(A) signal has a sequence which differs from naturally-occurring UTR elements. The sequence may be modified, for example by the deletion of ALU repeat sequences. Deletion of such ALU repeat sequences acts to reduce the possibility of homologous recombination between the expression cassette and genomic material in a transfected cell.
The plasmid preferably includes a promoter, a TATA box, a Cap site and a first intron and intron/exon boundary in appropriate relationship for expression of the coding sequence. The plasmid may also include a 5xe2x80x2 mRNA leader sequence inserted between the promoter and the coding sequence and/or an intron/5xe2x80x2 UTR from a chicken skeletal xcex1-actin gene. Also, the plasmid may have a nucleotide sequence which is the same as the nucleotide sequence of plasmid any of the plasmids described herein.
The plasmid may also include: (a) a first transcription unit containing a first transcriptional control sequence transcriptionally linked with a first 5xe2x80x2-untranslated region, a first intron, a first coding sequence, and a first 3xe2x80x2-untranslated region/poly(A) signal, wherein the first intron is between the control sequence and the first coding sequence; and (b) a second transcription unit containing a second transcriptional control sequence transcriptionally linked with a second 5xe2x80x2-untranslated region, a second intron, a second coding sequence, and a second 3xe2x80x2-untranslated region/poly(A) signal, wherein the second intron is between the control sequence and the second coding sequence.
Additionally, the in-line mixer may contain one or more other liquids, at least one of which is a formulating agent. The formulating agent preferably is a lipid, a peptide, a polymer or a small molecule such as europium. In one embodiment the formulating agent is also a protective, interactive, non-condensing compound.
In a preferred embodiment formulating agents are non-condensing polymers, oils and surfactants. These may be suitable for use as compounds which prolong the localized bioavailability of a nucleic acid: polyvinylpyrrolidones; polyvinylalcohols; propylene glycols; polyethylene glycols; polyvinylacetates; poloxamers (Pluronics) (block copolymers of propylene oxide and ethylene oxide, relative amounts of the two subunits may vary in different poloxamers); poloxamines (Tetronics); ethylene vinyl acetates; celluloses, including salts of carboxymethylcelluloses, methylcelluloses, hydroxypropylcellulose, hydroxypropylmethylcelluloses; salts of hyaluronates; salts of alginates; heteropolysaccharides (pectins); phosphatidylcholines (lecithins); miglyols; polylactic acid; polyhydroxybutyric acid. More preferably some of these compounds may be used as, and are considered protective, interactive, non-condensing compounds (PINC) and others as sustained release compounds, while some may be used in either manner under the respectively appropriate conditions.
By xe2x80x9cprolonging the localized bioavailability of a nucleic acidxe2x80x9d is meant that a nucleic acid administered to an organism in a composition comprising a formulating agent will be available for uptake by cells for a longer period of time than if administered in a composition without such a compound, for example when administered in a saline solution. This increased availability of nucleic acid to cells could occur, for example, due to increased duration of contact between the composition containing the nucleic acid and a cell or due to protection of the nucleic acid from attack by nucleases. The compounds which prolong the localized bioavailability of a nucleic acid are suitable for internal administration.
By xe2x80x9csuitable for internal administrationxe2x80x9d is meant that the compounds are suitable to be administered within the tissue of an organism, for example within a muscle or within a joint space, epidermally, intradermally or subcutaneously. Properties making a compound suitable for internal administration can include, for example, the absence of a high level of toxicity to the organism as a whole.
In another embodiment cationic condensing agents such as cationic lipids, peptides, or lipopetides, or for example, dextrans, chitosans, dendrimers, polyethyleneimine (PEI), or polylysine, may associate with the nucleic acid molecule and may facilitate transfection.
The PINC enhances the delivery of the nucleic acid molecule to mammalian cells in vivo, and preferably the nucleic acid molecule includes a coding sequence for a gene product to be expressed in the cell. In many cases, the relevant gene product is a polypeptide or protein. Preferably the PINC is used under conditions so that the PINC does not form a gel, or so that no gel form is present at the time of administration at about 30-40xc2x0 C. Thus, in these compositions, the PINC is present at a concentration of 30% (w/v) or less. In certain preferred embodiments, the PINC concentration is still less, for example, 20% or less, 10% or less, 5% or less, or 1% or. less. Thus, these compositions differ in compound concentration and functional effect from uses of these or similar compounds in which the compounds are used at higher concentrations, for example in the ethylene glycol mediated transfection of plant protoplasts, or the formation of gels for drug or nucleic acid delivery. In general, the PINCs are not in gel form in the conditions in which they are used as PINCs, though certain of the compounds may form gels under some conditions.
In connection with the protective, interactive, non-condensing compounds, the term xe2x80x9cnon-condensingxe2x80x9d means that an associated nucleic acid is not condensed or collapsed by the interaction with the PINC at the concentrations used in the compositions. Thus, the PINCs differ in type and/or concentration from such condensing polymers. Examples of commonly used condensing polymers include polylysine, and cascade polymers (spherical polycations).
The term xe2x80x9cprotectsxe2x80x9d or xe2x80x9cprotectivexe2x80x9d or xe2x80x9cprotectedxe2x80x9d as used herein refers to an effect of the interaction between such a compound and a nucleic acid such that the rate of degradation of the nucleic acid is decreased in a particular environment, thereby prolonging the localized bioavailability of the nucleic acid molecule. Such degradation may be due to a variety of different factors, which specifically include the enzymatic action of a nuclease. The protective action may be provided in different ways, for example, by exclusion of the nuclease molecules or by exclusion of water.
The term xe2x80x9cinteractivexe2x80x9d as used herein refers to the interaction between PINC""s and nucleic acid molecules and/or cell wall components. Preferably, PINC polymers are capable of directly interacting with moieties of nucleic acid molecules and/or cell wall components. These interactions can facilitate transfection by, for example, helping associate the nucleic acid molecule-PINC complex closely with the cell wall as a result of biochemical interactions between the PINC and the cell wall and thereby mediate transfection. These interactions may also provide protection from nucleases by closely associating with the nucleic acid molecule.
Also in connection with such compounds and an associated nucleic acid molecule, the term xe2x80x9cenhances the deliveryxe2x80x9d means that at least in conditions such that the amounts of PINC and nucleic acid is optimized, a greater biological effect is obtained than with the delivery of nucleic acid in saline. Thus, in cases where the expression of a gene product encoded by the nucleic acid is desired, the level of expression obtained with the PINC:nucleic acid composition is greater than the expression obtained with the same quantity of nucleic acid in saline for delivery by a method appropriate for the particular PINC/coding sequence combination.
In preferred embodiments of the above compositions, the PINC is polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), a PVP-PVA co-polymer, N-methyl-2-pyrrolidone (NM2P), ethylene glycol, or propylene glycol. In compositions in which a Poloxamer (Pluronics) is used, the nucleic acid is preferably not a viral vector, i.e., the nucleic acid is a non-viral vector.
In other preferred embodiments, the PINC is bound with a targeting ligand. Such targeting ligands can be of a variety of different types, including but not limited to galactosyl residues, fucosal residues, mannosyl residues, carntitine derivatives, monoclonal antibodies, polyclonal antibodies, peptide ligands, and DNA-binding proteins. The targeting ligands may bind with receptors on cells such as antigen-presenting cells, hepatocytes, myocytes, epithelial cells, endothelial cells, and cancer cells.
In connection with the association of a targeting ligand and a PINC, the term xe2x80x9cbound withxe2x80x9d means that the parts have an interaction with each other such that the physical association is thermodynamically favored, representing at least a local minimum in the free energy function for that association. Such interaction may involve covalent binding, or non-covalent interactions such as ionic, hydrogen bonding, van der Waals interactions, hydrophobic interactions, and combinations of such interactions.
While the targeting ligand may be of various types, in one embodiment the ligand is an antibody. Both monoclonal antibodies and polyclonal antibodies may be utilized.
The nucleic acid may also be present in various forms. Preferably the nucleic acid is not associated with a compounds(s) which alter the physical form, however, in other embodiments the nucleic acid is condensed (such as with a condensing polymer), formulated with cationic lipids, formulated with peptides, or formulated with cationic polymers.
In preferred embodiments, the protective, interactive non-condensing compound is polyvinyl pyrrolidone, and/or the plasmid is in a solution having between 0.5% and 50% PVP, more preferably about 5% PVP. The DNA preferably is at least about 80% supercoiled, more preferably at least about 90% supercoiled, and most preferably at least about 95% supercoiled.
The formulating agent may also protect the nucleic acid against freezing and may increase transfection rates. The effectiveness of the formulating agent in these two capacities may be seen for example in a comparison of gel electrophoresis results, run after thawing the solutions, of both the nucleic acid alone and with the formulating agent. The resulting gel of the nucleic acid with the formulating agent shows a much higher percentage of supercoiled plasmid. This higher percentage of supercoiled plasmid will therefore result in a higher transfection rate.
The term xe2x80x9cformulating agentxe2x80x9d as used herein refers to an agent that forms a complex with the nucleic acid. This molecular complex is associated with nucleic acid molecule in either a covalent or a non-covalent manner. The formulating agent should be capable of transporting nucleic acid molecules in a stable state and of releasing the bound nucleic acid molecules into the cellular interior. DNA extraction methods, methods of immunofluorescence, or well known reporter gene methods such as for example CAT, or LacZ containing plasmids, could be used in order to determine the transfection efficiency. The formulating agent should also be capable of being associated with nucleic acid molecules and lyophilized or freeze dried and rehydrated prior to delivery.
In addition, the formulating agent may prevent lysosomal degradation of the nucleic acid molecules by endosomal lysis. Furthermore, the formulating agent may allow for efficient transport of the nucleic acid molecule through the cytoplasm of the cell to the nuclear membrane and into the nucleus and provide protection.
In preferred embodiments, the formulating agent enhances the duration and/or intensity of expression of the desired gene.
By xe2x80x9cdurationxe2x80x9d is meant the amount of time a desired gene is expressed as measured, for example in months, weeks, days, hours, minutes and/or seconds.
By xe2x80x9cxe2x80x9cintensityxe2x80x9d is meant the rate at which a desired gene is expressed, for example mass or volume divided by time.
By xe2x80x9cexpressionxe2x80x9d is meant production of the encoded product, preferably by transcription and translation of the desired gene or nucleic acid sequence.
By xe2x80x9cdesired genexe2x80x9d is meant to refer to any gene or nucleic acid sequence encoding a product desired by the individual using the formulated nucleic acid complex. Examples of desired genes include CAT, and therapeutic agents (capable of at least partially reducing or preventing one or more symptoms of a disease) such as IL-2 and all other cytokines, as well as all intracellullar proteins (e.g., thymadine kinase).
The term xe2x80x9ccytokinesxe2x80x9d is meant to refer to the conventionally recognized group of immunogenic proteins such as IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-12, IL-18, TNF-xcex1, INF-xcex1, IFN-xcex1 and IFN-xcex3.
The formulating agent is preferably selected from the group consisting of: one or more polyvinyl pyrrolidones, one or more cationic lipids, one or more cationic lipids with neutral co-lipids, one or more liposomes, one or more peptides, and one or more lipopeptides.
Preferably the cationic lipid is DOTMA and the neutral co-lipid is cholesterol (chol). DOTMA is 1,2-di-O-octadecenyl-3-trimethylammonium propane, which is described and discussed in Eppstein et al., U.S. Pat. No. 4,897,355, issued Jan. 20, 1990, which is incorporated herein by reference. However, other lipids and lipid combinations may be used in other embodiments. A variety of such lipids are described in Gao and Huang, 1995, Gene Therapy 2:710-722, which is hereby incorporated by reference. Other cationic lipid delivery technology is described in Brigham, U.S. Pat. No. 5,676,954, issued Oct. 14, 1997, entitled xe2x80x9cMethod of In Vivo Delivery of Functioning Foreign Genesxe2x80x9d 216/012), incorporated herein by reference in its entirety, including any drawings.
As the charge ratio of the cationic lipid and the DNA is also a significant factor, in preferred embodiments the DNA and the cationic lipid are present in such amounts that the negative to positive charge ratio is between 1:0.1 and 1:10, preferably between 1:0.3 and 1:6, more preferably about 1:3. While preferable, it is not necessary that the ratio be 1:3. Thus, preferably the charge ratio for the compositions is between about 1:0.1 and 1:10, more preferably between about 1:0.3 and 1:6.
The term xe2x80x9ccationic lipidxe2x80x9d refers to a lipid which has a net positive charge at physiological pH, and preferably carries no negative charges at such pH. An example of such a lipid is DOTMA. Similarly, xe2x80x9cneutral co-lipidxe2x80x9d refers to a lipid which has is usually uncharged at physiological pH. An example of such a lipid is cholesterol.
Thus, xe2x80x9cnegative to positive charge ratioxe2x80x9d for the DNA and cationic lipid refers to the ratio between the net negative charges on the DNA compared to the net positive charges on the cationic lipid.
As the form of the DNA affects the expression efficiency, the DNA preferably is at least about 80% supercoiled, more preferably at least 90% supercoiled, and most preferably at least 95% supercoiled. The composition preferably includes an isotonic carbohydrate solution, such as an isotonic carbohydrate solution that consists essentially of about 10% lactose. In preferred embodiments, the composition the cationic lipid and the neutral co-lipid are prepared as a liposome having an extrusion size of between 200 and 900 nanometers, more preferably about 800 nanometers. Preferably the liposomes are prepared to have an average diameter of between about and 800 nm, more preferably between about 50 and 400 nm, still more preferably between about 75 and 200 nm, and most preferably about 100 nm. Microfluidization is the preferred method of preparation of the liposomes.
In a second aspect, the invention features a method of making an in-line mixer containing nucleic acid molecules as described above. The method involves the step of adding the nucleic acid molecules to the in-line mixer.
The angle between the two inlets preferably is between 45 and 300 degrees, more preferably between 90 and 240 degrees, and most preferably is between 120 and 180 degrees.
In a third aspect, the invention features a method of using an in-line mixer, involving the step of mixing the nucleic acid molecules with one or more other liquids (including emulsions, colloidal suspensions and other solutions) in the in-line mixer preferably where the liquids are continuously mixed. As the liquids are mixed, they preferably are being added to the Y-shaped configuration via a pump, adding the liquids in a continuous, syringe-like manner. The liquids may be added at a certain Reynolds number, preferably greater than 373, more preferably greater than 560, most preferably greater than 746, for optimal prevention of aggregation of the formulation. Non-Newtonian flow (Reynolds numbers greater than 1,000 with turbulence created in the mixing) may be possible.
By xe2x80x9cReynolds numberxe2x80x9d it is meant the quotient of the inertial forces in the apparatus divided by the viscous forces in the apparatus.
In a preferred embodiment, the liquids may be combined under conditions that produce a substantially homogenous mixture with particles of a preferred uniform size preferably less than or equal to 100 nm, more preferably less than or equal to 75 nm, most preferably less than or equal to 50 nm.
By xe2x80x9cuniform sizexe2x80x9d it is meant that most of the particles in the complex are approximately the same size and are less than the specified value. To be of uniform size, the particles do not have to be the identical size, but are uniformly smaller than the expected size. In another embodiment, the liquid of nucleic acid molecules may be combined with one, two, three or more other liquids.
In a fourth aspect, the invention features a co-lyophilized complex produced by the co-lyophilization of a nucleic acid molecule in a vector with a formulating agent that protects the nucleic acid molecule against freezing and increases transfection rates.
By xe2x80x9cco-lyophilizedxe2x80x9d it is meant the process by which the mixture of the two or more liquids is freeze-dried to protect against the instability of the nucleic acid in solution. Dehydration preferably take place while the product is in a frozen state and under a vacuum. The basic theory, equipment and methodology relting to lyopholiztion of other materials is well known and those skilled in the art therefore could readily select the appropriate parameters for effective lyophilization. See, FTS Systems, Basic Theory of Freeze Drying/Lyophilization, Product Support Information, Bulletin #1:1-19; Nail et al., Develop. Biol. Standard., Vol. 74, pp. 137-151 (1991); Jennings, et al. DandCl, pp 43-52 (1980); Reiter, American Laboratory, xe2x80x9cSignificant Design Changes in Laboratory/Research Freeze Dryersxe2x80x9d, (1991); Thompson et al., InTech, xe2x80x9cEvolving Instrumentation for Freeze-Dryingxe2x80x9d, (1995); Jennings, Journal of Parenteral Science and Technology, Vol. 42:118-121 (1988); Jennings, MDandDI, pp 49-56 (1980); Livesey et al., Journal of Parenteral Science and Technology, Vol. 41, No. 5;169-171 (1987); Jennings, Journal of Parenteral Science and Technology, pp. 95-97 (1986); Leebron et al., Journal of Parenteral Science and Technology, Vol. 35, No. 3:100-105 (1981); Williams et al., Journal of Parenteral Science and Technology, Vol. 40, No. 4: 135-141 (1986); Roy et al., Research Article, Vol. 43, No. 2:60-66 (1989); Armstrong, Journal of Parenteral Drug Association, Vol. 34, No. 6: 473-483 (1980); Jennings, Journal of the Parental Drug Association, Vol. 34, No. 1:62-69 (1980) all of which are incorporated herein by reference in their entirety, including any drawings
In a preferred embodiment, the formulating agent may be a protective, interactive, non-condensing compound. In especially preferred embodiments the formulating agent may be pre-neutralized polyvinyl pyrrolidone, preferably in a concentration of at least 2.5% or a molecular weight of at least 80 kDa. The formulating agent may also be polyvinyl alcohol present in a weight to weight ratio with the formulating agent of about 1 to 17. In a preferred embodiment the complex will be present in a solution having a pH of preferably 3.5 to 9.0, more preferably 6.5 to 8.0 in order to maximize nucleic acid expression.
In other preferred embodiments, the complex may also include an antimicrobial agent, an anti-oxidant, a buffer, or a cryoprotectant.
By xe2x80x9cantimicrobial agentxe2x80x9d it is meant any chemical or compound which is capable of destroying or inhibiting the growth of microorganisms. In a preferred embodiment, the antimicrobial agent may be Benzalkonium chloride, Benzyl alcohol, Chlorocresol, Phenylmercuric nitrate, oracetate.
By xe2x80x9canti-oxidantxe2x80x9d it is meant a chemical compound or substance that inhibits oxidation. In a preferred embodiment, the anti-oxidant may be Ascorbic acid, Butylhydroxyanisole (BHA), Cysteine, Sodium bisulfate, or Glutathione.
By a xe2x80x9cbufferxe2x80x9d it is meant a substance that minimizes change in the pH of a solution when an acid or base is added to the solution. In a preferred embodiment, the buffer may be Acetic acid and salt, Succinic acid and borax, Formate and HCl, or Na-citrate buffer.
By xe2x80x9ccryoprotectantxe2x80x9d it is meant any chemical or compound that will serve to protect a substance during freezing. In a preferred embodiment, the cryoprotectant may be lactose, sucrose, mannitol, trehalose, or polyvinyl pyrrolidone.
In a fifth aspect the invention features the making of a co-lyophilized complex preferably made by combining a liquid of nucleic acid molecules and a liquid formulating agent. Preferably the two liquids are passed through an in-line mixer and continuously mixed. The liquids may be added at a certain Reynolds number, preferably greater than 373, more preferably greater than 560, most preferably greater than 746, for optimal prevention of aggregation of the formulation. In a preferred embodiment, the liquids may be combined under conditions that produce a substantially homogenous mixture with particles of a preferred uniform size preferably less than or equal to 100 nm, more preferably less than or equal to nm, most preferably less than or equal to 50 nm. In another embodiment, the liquid of nucleic acid molecules may be combined with one, two, three or more other liquids.
In a sixth aspect, the invention features a method of using the complex resulting from the in-line mixing, by re-hydration.
By xe2x80x9cre-hydrationxe2x80x9d it is meant to cause the complex to take up fluid, preferably a pharmaceutically acceptable solution such as isotonic saline, buffer or other solution.
In a seventh aspect, the invention features a method of using the complex in the treatment or prevention of a disorder by administration of the complex, in any of the above forms, to a patient in need of such treatment.
By xe2x80x9cadministrationxe2x80x9d it is meant the route of introduction of a vector or carrier of DNA into the body. Administration may be intravenous, intramuscular, topical, oral,.or by gene gun or hypospray instrumentation. It can be directly to a target tissue or through systemic delivery. Administration will include a variety of methods, such as direct gene transfer into skin tissue by liposomes, proteoliposomes, calcium phosphate-coprecipitated DNA, DNA coupled to macromolecular complexes, DNA transporters, DNA coded to microprojectiles, coded plasmids, direct microinjection, as well as skin grafts. Direct gene transfer of vectors can be administered by direct microinjection, needle-free injection (see, Barry et al., xe2x80x9cNeedle-Free Injection of Formulated Nucleic Acid Moleculesxe2x80x9d, U.S. Patent Application, Ser. No. 60/069,754, filed Dec. 6, 1997, incorporated herein by reference in its entirety, including any drawings), sonoporation, electroporation (see, MaClaughlin et al., xe2x80x9cFormulations for Electroporationxe2x80x9d, U.S. Patent Application, Ser. No. 60/088,691, filed, Jun. 8, 1998, incorporated herein by reference in its entirety, including any drawings), liposomes, proteoliposomes, calcium phosphate-coprecipitation, skin grafts, retroviral vectors, DNA coupled to macromolecular complexes, DNA transporters and microprojectiles. Routes of administration include intramuscular, aerosol, oral, topical, systemic, ocular, intraperitoneal and/or intrathecal. See, e.g., Woo, et al, International Application No. PCT/US93/02725, filed Mar. 19, 1993, International Publication No. WO 93/18759, published Sep. 30, 1993, entitled xe2x80x9cA DNA Transporter System and Method of Usexe2x80x9d hereby incorporated by reference in its entirety, including any drawings and the section on administrative in the detailed description section below.
By xe2x80x9ctreatxe2x80x9d it is meant administration of the nucleic acid as described herein so as to deliver a desired nucleic acid to a cell or tissue for the purposes of expression of the nucleic acid by the cell or tissue. Cell or tissue types of interest may include, but are not limited to: liver, muscle, lung, endothelium, joints, skin, bone, tumors and blood.
By xe2x80x9cpreventxe2x80x9d it is meant to stop or hinder the disorder from occurring by advance action, the delivery of the nucleic acid to the cell, to inhibit the cell from completing the action it would undergo to express such disorder.
In preferred embodiments, the method involves providing a therapeutically effective amount of the complex.
A xe2x80x9ctherapeutically effective amountxe2x80x9d of a composition is an amount which is sufficient to cause at least temporary relief or improvement in a symptom or indication of a disease or condition. Thus, the amount is also sufficient to cause a pharmacological effect. The amount of the composition need not cause permanent improvement or improvement of all symptoms or indications. A therapeutically effective amount of a cancer therapeutic would be one that reduces overall tumor burden in the case of metastatic disease (i.e., the number of metasteses or their size) or one that reduces the mass of the tumor in localized cancers.
The disorder being treated may be localized or systemic disease or condition
A xe2x80x9clocalizedxe2x80x9d disease or condition refers to those in which there is specific nerve or muscle damage or atrophy to a defined and limited area of the body. A specific example is disuse atrophy.
A xe2x80x9csystemicxe2x80x9d disease or condition refers to those which relate to the entire organism, or is widely distributed at a number of locations within the body. Examples include growth disorders, neuropathies, and muscular dystrophy.
In an eighth aspect, the invention features a method of delivering the complex by administration, in any of the above forms, to an organism, preferably an animal.
By xe2x80x9cdeliveryxe2x80x9d or xe2x80x9cdeliveringxe2x80x9d is meant transportation of nucleic acid molecules to desired cells or any cells. The nucleic acid molecules may be delivered to multiple cell lines, including the desired target. Delivery results in the nucleic acid molecules coming in contact with the cell surface, cell membrane, cell endosome, within the cell membrane, nucleus or within the nucleus, or any other desired area of the cell from which transfection can occur within a variety of cell lines which can include but are not limited to; tumor cells, epithelial cells, Langerhan cells, Langhans"" cells, littoral cells, keratinocytes, dendritic cells, macrophage cells, kupffer cells, muscle cells, lymphocytes and lymph nodes.
Preferably, the vector comes into contact with the preferred target cell after administration. Administration as noted above, may involve needle injection into cells, tissues, fluid spaces, or blood vessels, electroporation, transfection, hypospray,. iontophoresis, particle bombardment, or transplantation of cells genetically modified ex vivo. Examples of administration include intravenous, intramuscular, aerosol, oral, topical, systemic, ocular, intraperitoneal and/or intrathecal.
The preferred means for administration of vectors described above involves the use of formulations for delivery to the target cell in which the vector is associated with elements such as lipids, proteins, carbohydrates, synthetic organic compounds, or in-organic compounds which enhance the entry of the vector into the nucleus of the target cell where gene expression may occur. A particular example is polyvinyl pyrrolidone(PVP).
The term xe2x80x9cformulationxe2x80x9d as used herein refers to non-genetic material combined with the vector in a solution, suspension, or colloid which enhances the delivery of the vector to a tissue, uptake by cells within the tissue, intracellular trafficking through the membrane, endosome or cytoplasm into the nucleus, the stability of the vector in extracellular or intracellular compartments, and/or expression of genetic material by the cell.
In a preferred embodiment of the present invention the vector and formulation comprises a nanoparticle which is administered as a suspension or colloid. The formulation can include lipids, proteins, carbohydrates, synthetic organic compounds, or inorganic compounds. Examples of elements which are included in a formulation are lipids capable of forming liposomes, cationic lipids, hydrophilic polymers, polycations (e.g. protamine, polybrine, spermidine, polylysine), peptide or synthetic ligand recognizing receptors on the surface of the target cells, peptide or synthetic ligand capable of inducing endosomal-lysis, peptide or synthetic ligand capable of targeting materials to the nucleus, gels, slow release matrices, salts, carbohydrates, nutrients, or soluble or insoluble particles as well as analogues or derivatives of such elements. This includes formulation elements enhancing the delivery, uptake, stability, and/or expression of genetic material into cells. This list is included for illustration only and is not intended to be limiting in any way.
The term xe2x80x9corganismxe2x80x9d as used herein refers to common usage by one of ordinary skill in the art. The organism can include; micro-organisms, such as yeast or bacteria, plants, birds, reptiles, fish or mammals. The organism can be a companion animal or a domestic animal. Preferably the organism is a mammal and is therefore any warm blooded organism. More preferably the mammal is a human.
The term xe2x80x9ccompanion animalxe2x80x9d as used herein refers to those animals traditionally treated as xe2x80x9cpetsxe2x80x9d such as for example, dogs, cats, horses, birds, reptiles, mice, rabbits, hamsters, and the like.
The term xe2x80x9cdomestic animalxe2x80x9d as used herein refers to those animals traditionally considered domesticated, where animals such as those considered xe2x80x9ccompanion animalsxe2x80x9d are included along with animals such as, pigs, chickens, ducks, cows, goats, lambs, and the like.
In another embodiment the method results in an immune response, preferably a humoral immune response targeted for the protein product encoded by the nucleic acid molecule, such as an antibody response. In other situations the immune response preferably is a cytotoxic T-lymphocyte response.
The term xe2x80x9cimmune responsexe2x80x9d as used herein refers to the mammalian natural defense mechanism which can occur when foreign material is internalized. The immune response can be a global immune response involving the immune system components in their entirety. Preferably the immune response results from the protein product encoded by the formulated nucleic acid molecule. The immune response can be, but is not limited to; antibody production, T-cell proliferation/differentiation, activation of cytotoxic T-lymphocytes, and/or activation of natural killer cells. Preferably the immune response is a humoral immune response. However, as noted above, in other situations the immune response, preferably, is a cytotoxic T-lymphocyte response.
The term xe2x80x9chumoral immune responsexe2x80x9d refers to the production of antibodies in response to internalized foreign material. Preferably the foreign material is the protein product encoded by a formulated nucleic acid molecule.
In a preferred embodiment the method results in enhanced transfection of cells. The enhanced transfection can be measured by transfection reporter methods commonly known in the art such as, for example, assays for CAT gene product activity, or LacZ gene product activity, and the like.
The term xe2x80x9ceffective amountxe2x80x9d as used herein refers to sufficient vector administered to humans, animals or into tissue culture to produce the adequate levels of protein or RNA. One skilled in the art recognizes that the adequate level of protein or RNA will depend on the use of the particular vector. These levels will be different depending on the type of administration and treatment or vaccination.
The methods for treating diseases as disclosed herein includes treatment with biological products (specifically proteins as defined above) in which the disease being treated requires the protein to circulate through the body from the general circulation. For example, disorders which might be treated by the present invention include osteoporosis by expression of GHRH or its binding proteins. The selection of the appropriate protein to treat various diseases will be apparent to one skilled in the art.
In treating disease, the present invention provides a means for achieving: (1) sufficiently high levels of a particular protein to obtain a therapeutic effect; (2) controlled expression of product at levels which are sufficient for therapeutic effect and lower than the toxic levels; (3) controlled expression in certain tissues in order to obtain reproducible pharmacokinetics and levels of gene expression; and (4) delivery using clinically and pharmaceutically acceptable means of administration and formulation rather than transplantation of genetically engineered and selected cells.
In a ninth aspect, the invention features a homogenous mixture that is a plurality of any of the above-specified complexes where each complex has particles of substantially uniform size. In a preferred embodiment each of the complexes will have particles which are approximately spherical and which have a diameter of preferably 500 nm or less, more preferably 200 nm or less, -most preferably 100 nm or less.
In another aspect the invention features a kit. The kit includes a formulated nuclic acid complex of the invention, preferably in a container and/or with instructions explaining how to deliver the formulated nucleic acid molecules.
Thus, the xe2x80x9ccontainerxe2x80x9d can include instructions furnished to allow one of ordinary skill in the art to use the formulated nucleic acid molecules. The instructions will furnish steps to use the formulated nucleic acid molecules. Additionally, the instructions may include methods for testing the formulated nucleic acid molecules that entail establishing if the formulated nucleic acid molecules are damaged. The kit may also include notification of an FDA approved use and instructions.
In another aspect, the invention features a method for making a kit. Preferably the method involves the step of combining a container with a nucleic acid formulated with a formulating agent and/or instructions.
In another aspect the invention features cells transfected or transformed with the formulated nucleic acid complex of the invention and methods of making and using the same.
The term xe2x80x9ctransfectionxe2x80x9d as used herein refers to the process of introducing DNA (e.g., formulated DNA expression vector) into a cell, thereby, allowing cellular transformation. Following entry into the cell, the transfected DNA may: (1) recombine with that of the host; (2) replicate independently as a plasmid or temperate phage; or (3) be maintained as an episome without replication prior to elimination.
As used herein, xe2x80x9ctransformationxe2x80x9d relates to transient or permanent changes in the characteristics (expressed phenotype) of a cell induced by the uptake of a vector by that cell. Genetic material is introduced into a cell in a form where it expresses a specific gene product or alters the expression or effect of endogenous gene products.
Transformation of the cell may be associated with production of a variety of gene products including protein and RNA. These products may function as intracellular or extracellular structural elements, ligands, hormones, neurotransmitters, growth regulating factors, enzymes, chemotaxins, serum proteins, receptors, carriers for small molecular weight compounds, drugs, immunomodulators, oncogenes, cytokines, tumor suppressors, toxins, tumor antigens, antigens, antisense inhibitors, triple strand forming inhibitors, ribozymes, or as a ligand recognizing specific structural determinants on cellular structures for the purpose of modifying their activity. This list is only an example and is not meant to be limiting.
The summary of the invention described above is non-limiting and other features and advantages of the invention will be apparent from the following detailed description of the preferred embodiments, as well as from the claims.